One of the advances in modern biotechnology is the large-scale production of proteins or protein-containing gene products of interest. Bacterial host cells are often used for the production of proteins of interest because of their ability to be easily fermented in large volumes. One crucial disadvantage of numerous bacterial systems is their use of rare codons, which can be very different from the codon preference of a gene being expressed. Rare codons in bacteria are typically associated with low level expression of tRNAs bearing the complimentary anticodon. As a result, the presence of rare codons in the gene being expressed can lead to significantly reduced expression of the desired product in the bacterial host cell due to a depletion of pools of rare tRNAs.
In order to increase expression in bacterial systems, one approach has been to change rare codons in the gene being expressed to a codon occurring more frequently in the host cell that codes for the same amino acid. See U.S. Pat. No. 6,821,755. Other examples of modification of a gene of interest to replace rare codons with more favored codons may be found in Prapunwattana, et al., Mol Biochem. Parasitol. 83:93-106 (1996) and Pan, et al., Nucleic Acids Res. 27:1094-1103 (1999). This approach is limited, however, by the need to perform perhaps multiple steps of mutagenesis on the gene of interest. In addition, the process only leads to increased expression of one gene. The entire process of replacing rare codons for abundant codons must be repeated on every gene of interest.
Another approach to increase expression in bacterial systems has been to introduce into the host cells a plasmid that contains genes coding for cognate tRNAs of the rare codons. For example, several plasmids have been constructed to increase the expression of rare tRNAs. U.S. Pat. No. 6,270,988 describes plasmids containing genes for rare tRNA molecules for AGG and AGA codons for arginine. Baca, et al. Int'l. J. Parasit. 30:113, 118 (2000) describe a plasmid derived from pACYC 184 encoding tRNAs for arginine, isoleucine, and glycine: argU (AG(A/G)), ileX (AUA), and glyT (GG(A/G)). Commercially cells are also available that contain plasmids providing additional copies of rare tRNAs. Using plasmids for expressing rare tRNAs is limited by the need for a selectable marker, which increases production costs. Furthermore, the typically used high copy number plasmids can lead to genetic instability in the host cell. The art continues to need, therefore, an efficient system for producing proteins with rare codons. The present invention satisfies this need and others.